Hi there,
I want to generate retroviral producer cell line using Phoenix eco cells for transfection and E86 cells for transduction. My construct is about 9.2 kb but the insert part (from 3' of 5'LTR to 3' of 3'LTR) is about 5.5kb. The size of my construct is not small but I think it can be up to 8kb so it should be ok but my several attempts have failed . Could you please help me and advise me?
Thank you all
Hi Niloofar,
You failed to generate retroviral producer cell line or your large vector?
In my expereince, we already produce with success lentiviral vector > 10kb with a protocol of 293T tri-transfection, so your challenge seems achievable.with the good conditions and protocol.
Regards
Nicolas
Hello, You don't mention where your protocol fails. Do you see your transfected 293T cells expressing your protein of interest? Do you see your cells from syncytia? Are your control vectors working in transduction?
Your retroviral construct does not appear to be too big to form viral particles. I have previously made virus from 14kb constructs in Eco cells. However, the viral titre decreases as the size of your construct increases. Furthermore, remember that these retroviruses can only transduce dividing cells and that the ecotropic pseudotype is not as robust as VSVG so they are more sensitive to freeze/thaw. In the past I've only used fresh virus.
Also note that for some cell types, especially primary cells, you might need to concentrate your virus (e.g. with PEG or ultracentrifugation) to get sufficient titre.
Hi Nicolas and Qiang
Thank you so much for your answers. I could transfect my Phoenix eco cells as I could see GFP glowing under microscope but I couldn't transduce E86 cells. I think I have problem with virus packaging. I always use fresh virus I haven't freeze/thaw them. I also use dividing E86 cells. I usually seed 50,000 and 200,000 E86 cells on the day before transduction.
Hi Niloofar,
Could you please describe your expression cassette please.
Best
Nicolas
Hi Nicolas,
It has: 5'UTR, MESV psi, gag (truncated), my genes linked to each other by IRES and P2A, 3'UTR on one direction and M13 rev, lac operator, lac promoter, CAP binding site, ori, AmpR, AmpR promoter on another direction.
Thank you so much,
Niloo
Hi Niloofar,
Ok, I see some possible explanations
I don't know if GFP is included in your IRES system but I already saw in this case GFP expression in transfection and no GFP in transduction although the other transgene was, this is a classical trouble observed with IRES. I tink it is better to have different 2A linkers if you want express more than two transgenes with one vector.
Your IRES system and 2A system are they in opposite direction? Have you an internal polyA sequence in sens in the vector genome? If it is the case, this can highly decrease the titer of the vector, depending the context.
Article An Internal Polyadenylation Signal Substantially Increases E...
regards
Nicolas
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